© 2013. Published by The Company of Biologists Ltd | Development (2013) 140, 4709-4718 doi:10.1242/dev.099028

RESEARCH ARTICLE

Pax9 regulates a molecular network involving Bmp4, Fgf10, Shh signaling and the Osr2 to control Jing Zhou1, Yang Gao2, Yu Lan1, Shihai Jia1 and Rulang Jiang1,2,*

ABSTRACT Hilliard et al., 2005; Li and Ding, 2007; Liu et al., 2008; Welsh and Cleft palate is one of the most common birth defects in humans. O’Brien, 2009). For example, the short stature 2 (Shox2), Whereas knockout studies in mice have shown that both the Msh homeobox 1 (Msx1) and fibroblast growth factor 10 (Fgf10) Osr2 and Pax9 transcription factors are essential regulators of are preferentially expressed in the anterior palatal palatogenesis, little is known about the molecular mechanisms mesenchyme (Alappat et al., 2005; Rice et al., 2004; Yu et al., 2005; involving these transcription factors in palate development. We report Zhang et al., 2002), whereas the BarH-like homeobox 1 (Barx1), here that Pax9 plays a crucial role in patterning the anterior-posterior meningioma 1 (Mn1), mesenchyme homeobox 2 (Meox2) and T-box axis and outgrowth of the developing palatal shelves. We found that transcription factor 22 (Tbx22) genes are preferentially expressed in tissue-specific deletion of Pax9 in the palatal mesenchyme affected the posterior palatal mesenchyme (Barlow et al., 1999; Li and Ding, Shh expression in palatal epithelial cells, indicating that Pax9 plays 2007; Liu et al., 2008), with the AP gene expression boundary a crucial role in the mesenchyme-epithelium interactions during coinciding with the first formed palatal ruga (Welsh and O’Brien, palate development. We found that expression of the Bmp4, Fgf10, 2009). Moreover, anterior and posterior palatal mesenchyme cells Msx1 and Osr2 genes is significantly downregulated in the exhibit distinct responses to epithelial signals. Exogenous Bmp4 developing palatal mesenchyme in Pax9 mutant embryos. induced Msx1 mRNA expression in the anterior, but not posterior, Remarkably, restoration of Osr2 expression in the early palatal palatal mesenchyme, whereas Fgf8 induced Pax9 mRNA expression mesenchyme through a Pax9Osr2KI allele rescued posterior palate in the posterior, but not anterior, palatal mesenchyme (Hilliard et al., morphogenesis in the absence of Pax9 function. Our data 2005). In addition, the anterior palatal epithelium has the unique indicate that Pax9 regulates a molecular network involving the Bmp4, ability to induce Shox2 mRNA expression in the palatal Fgf10, Shh and Osr2 pathways to control palatal shelf patterning and mesenchyme (Yu et al., 2005). These data indicate that the morphogenesis. developing palate is patterned along the AP axis by restricted gene expression in both the epithelium and mesenchyme. KEY WORDS: Cleft palate, Bmp4, Osr2, Pax9, Shh, Mouse Recent studies have shown that the anteriorward outgrowth of palatal shelves is associated with periodic formation of palatal rugae INTRODUCTION and is controlled by reciprocal epithelial-mesenchymal crosstalk The mammalian secondary palate initiates from the oral side of the along the AP axis (Lan and Jiang, 2009; Pantalacci et al., 2008; maxillary processes as a pair of outgrowths that grow vertically Welsh and O’Brien, 2009). The palatal rugae are the thickened down the sides of the developing . At a precise developmental epithelial ridges on the oral side of the secondary palate, and are stage, the palatal shelves re-orient to the horizontal position above formed in a defined sequence during palate outgrowth (Welsh and the dorsum of the tongue, and grow towards and subsequently fuse O’Brien, 2009). Palatal rugae express high levels of Shh and act as with each other at the midline. In addition, the palatal shelves fuse signaling centers involved in epithelial-mesenchymal interactions anteriorly with the , which is derived from the required to coordinate palate outgrowth and patterning (Lan and embryonic medial nasal processes, to form the intact roof of the oral Jiang, 2009; Rice et al., 2006; Welsh and O’Brien, 2009). Shh from cavity. Disturbances of the growth, elevation or fusion of the palatal palatal epithelial cells stimulates cell proliferation and the expression shelves could result in cleft palate, one of the most common birth of Fgf10 in the developing palatal mesenchyme (Lan and Jiang, defects in humans (Bush and Jiang, 2012; Ferguson, 1988; Gritli- 2009), and Shh and Fgf10 function in a positive-feedback loop that Linde, 2007). drives the outgrowth of the palatal shelves (Bush and Jiang, 2012; The developing palatal shelves are composed of neural crest- Lan and Jiang, 2009; Rice et al., 2004). In addition, cross-regulation derived mesenchyme (Ito et al., 2003) covered by a thin layer of oral of the Shh and bone morphogenetic protein (Bmp) signaling epithelium, and exhibit distinct stereotyped shapes along the pathways has also been detected in the developing palate (Baek et anterior-posterior (AP) axis (Bush and Jiang, 2012). Recent studies al., 2011; Lan and Jiang, 2009; Zhang et al., 2002). have demonstrated that many genes are differentially expressed We have reported previously that mice homozygous for a targeted along the AP axis in the developing palate (Baek et al., 2011; null mutation in the Osr2 gene had complete cleft palate (Lan et al., 2004). Osr2 encodes a zinc-finger protein with extensive sequence similarity to the Odd-skipped family transcription 1Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA. 2Center for Oral Biology and Department of Biomedical Genetics, University of factors that regulate multiple developmental processes during Rochester, Rochester, NY 14642, USA. embryogenesis and tissue morphogenesis (Coulter and Wieschaus, 1988; Green et al., 2002; Hao et al., 2003; Hart et al., 1996; Lan et *Author for correspondence ([email protected]) al., 2001; Saulier-Le Dréan et al., 1998; Wang and Coulter, 1996). −/−

Received 14 May 2013; Accepted 2 September 2013 Osr2 mutant mice exhibited domain-specific defect in palatal DEVELOPMENT

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Fig. 1. Pax9 mRNA expression during palate development. (A,B) Whole- mount in situ hybridization detection of Pax9 mRNA in the developing palatal shelves in E12.5 (A) and E13.5 (B) mouse embryos. (C-E) Frontal sections through distinct planes along the anterior-posterior axis of the E13.5 palatal shelves showing Pax9 mRNA expression pattern in the anterior (C), middle (D) and posterior (E) regions. Dotted white lines marked c-e in B indicate the section planes for C-E, respectively. m, molar tooth germ; p, palatal shelf. mesenchyme cell proliferation and delay in palatal shelf elevation (Lan et al., 2004). In addition to cleft palate, Osr2−/− mutant mice developed supernumerary teeth lingual to their molar teeth (Zhang et al., 2009). Osr2 mRNA expression exhibited a lingual-to-buccal gradient, complementary to that of Bmp4, in the mesenchyme surrounding the early developing tooth buds and patterns the tooth developmental field by suppressing the propagation of mesenchymal odontogenic activity driven by the Msx1-Bmp4 positive-feedback loop (Jia et al., 2013; Zhang et al., 2009). Fig. 2. Histological analysis of palate developmental defects in Paired-box gene 9, also known as Pax9, is a member of the del/del transcription factor family characterized by the Paired-class DNA- Pax9 mutant mouse embryos. Representative frontal sections from the anterior, middle and posterior regions of the developing palatal shelves binding domain (Stapleton et al., 1993). Pax9-deficient mice die are shown. (A-F) E13.5 control (A-C) and mutant (D-F) littermates. shortly after birth, exhibiting complete cleft palate (Peters et al., (G-L) E14.5 control (G-I) and mutant (J-L) littermates. (M-R) E15.5 control 1998). Little is known about the molecular and cellular mechanisms (M-O) and mutant (P-R) littermates. m1, first molar tooth germ; p, palatal involving Pax9 in palate development, however. We recently shelf; t, tongue. generated a mouse strain (Pax9fneo/+) that allows the Cre/loxP- mediated inactivation of the Pax9 gene and subsequent activation of the nasal half of the palatal mesenchyme in the anterior region transgenic Osr2 expression from the endogenous Pax9 (Zhou (Fig. 1C,E). et al., 2011). We report here that Pax9 is required for maintenance of Osr2 expression in the developing palatal mesenchyme and that Defects in palatal shape and AP patterning in Pax9 mutant expression of Osr2 from the Pax9 locus partially rescued palate mice morphogenesis in Pax9-null mutant mice. We recently generated mice carrying a deletion of the first two exons of the Pax9 gene (Pax9del) and showed that Pax9del/del mutant RESULTS mice displayed similar developmental defects, as reported Pax9 exhibits differential expression patterns along the AP previously in another Pax9-deficient mouse strain, including cleft axis of the developing palate palate and tooth developmental arrest (Peters et al., 1998; Zhou et To study the mechanism by which Pax9 regulates palate al., 2011). Detailed histological analysis of Pax9del/del mutant development, we first examined the expression patterns of Pax9 embryos showed that the mutant palatal shelves had an aberrant during palate development by whole-mount and section in situ shape and lacked the lateral indentation between the maxillary molar hybridization. From E12.5 to E13.5, when the palate shelves grew tooth germ and the palatal shelf at E13.5, in comparison with the vertically, Pax9 exhibited a posterior-to-anterior gradient expression wild-type littermates (Fig. 2A-F). At E14.5, the palatal shelves had pattern in the palatal mesenchyme (Fig. 1A,B). Moreover, whereas elevated to the horizontal position above the developing tongue and Pax9 mRNAs were expressed throughout the oral-nasal axis of the initiated fusion at the midline in the control embryos (Fig. 2G-I). By palatal mesenchyme in the posterior region (Fig. 1E), Pax9 mRNA contrast, the mutant embryos exhibited delay in palatal shelf

expression exhibited apparently lower levels in the oral half than in elevation (Fig. 2J-L). By E15.5, the midline epithelial seam between DEVELOPMENT

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expression was much weaker and not distinctly localized in discrete rugae structures in the middle region of the palatal shelves at E13.5 in the mutant embryos (Fig. 4G). By E14.5, the Shh-expressing rugae structures were better demarcated but there were fewer rugae and the distance between R1 and R5 was significantly shorter in the mutant embryos than in the control littermates (compare Fig. 4D with 4H), indicating impairment in anteriorward outgrowth of the palatal shelves in the mutant embryos. In addition, the punctate Shh expression pattern observed in the developing sensory papilla overlying the posterior soft palate was absent in the Pax9del/del mutant embryos (Fig. 4D,H). The mutant embryos also exhibited reduced Shh expression in the primary palate, in comparison with the control littermates (Fig. 4D,H). Pax9 was expressed in the palatal mesenchyme, as well as in the posterior palatal epithelium (Fig. 1). To investigate whether defects Fig. 3. Scanning electron microscopy analysis of palate developmental in Shh expression were due to loss of Pax9 function in the defects in Pax9del/del mutant mice. (A-D) Oral view of the developing palate del/del epithelium, we inactivated Pax9 specifically in neural crest-derived in wild-type (A,B) and Pax9 mutant (C,D) embryos at E13.5. Rectangle fneo in D marks the region of deficiency in primary palate. mesenchymal cells by crossing the Pax9 mice with Wnt1-Cre transgenic mice (Danielian et al., 1998). We found that the Pax9fneo/delWnt1-Cre mutant embryos showed similar defects in Shh the palatal shelves had mostly disappeared in the control embryos mRNA expression to the Pax9del/del mutant embryos in both the (Fig. 2M-O). By contrast, the mutant palatal shelves remained primary and secondary palate (compare Fig. 5 with Fig. 4). These separate from each other (Fig. 2P-R). In addition to the lack of results suggest that Pax9 plays an important role in regulating the palatal fusion, scanning electron microscopy studies showed that the mesenchymal-epithelial interactions during palate development. mutant palatal shelves had defects in formation of the palatal rugae, In addition to defects in the anteriorward outgrowth of the palate which are the thickened epithelial ridges on the oral side of the shelves, we found that the anterior-posterior boundary of the palatal palatal shelves, and deficiency in primary palate outgrowth mesenchyme was disturbed in the Pax9del/del mutant embryos. As (Fig. 3A-D). shown in Fig. 6, expression of Shox2 was restricted anterior to R1, Recent studies showed that the palatal rugae express high levels whereas Barx1 expression was restricted posterior to R1 in the of Shh mRNAs and are formed in a defined temporal sequence E13.5 wild-type palate (Fig. 6A,C). By contrast, both the posterior during palate outgrowth (Baek et al., 2011; Pantalacci et al., 2008; boundary of Shox2 expression and the anterior boundary of Barx1 Welsh and O’Brien, 2009). Our whole-mount in situ hybridization expression appeared diffuse and anteriorly shifted in the Pax9del/del analyses confirmed that the Pax9del/del mutant embryos had defects mutant palate (Fig. 6B,D). Taken together, these results indicate that in palatal rugae formation. At E12.0, when two pairs of clearly deletion of Pax9 resulted in disruption of the AP boundary of the defined rugae had formed in wild-type embryos (Fig. 4A), the palatal mesenchyme, which correlated with the misshapen palatal Pax9del/del mutant embryos showed diffuse Shh mRNA expression in shelves and defects in expansion of the palatal shelves along the AP the palatal epithelium and lacked clearly demarcated rugae axis in the Pax9del/del mutant embryos. structures (Fig. 4E). From E12.5 to E14.5, the palatal rugae formed sequentially in a defined sequence as the palatal shelves grew Pax9 acts upstream of Bmp4, Fgf10, Msx1 and Osr2 in the rostrally (Fig. 4B-D). In the mutant palate, although two pairs of palatal mesenchyme strong Shh-expressing rugae were detected in the anterior most The defects in Shh expression in the palatal epithelium in both the region, corresponding to R2 and R3 in the control palate, Shh Pax9del/del and Pax9fneo/del;Wnt1-Cre mouse embryos indicate that

Fig. 4. Analysis of palatal ruga formation and anteriorward palatal outgrowth in Pax9del/del mutant embryos. (A-H) Whole-mount in situ hybridization detection of patterns of Shh mRNA expression in the developing palate in wild-type control (A-D) and Pax9del/del mutant (E-H) embryos from E12.0 to E14.5. Strong Shh mRNA expression marks the palatal rugae, which increase in number from two at E12.0 to seven pairs by E14.5 in the wild-type embryos (A-D), whereas the mutant had formed only five pairs by E14.5 (E-H). p, palatal shelf; pp, primary palate; sp, sensory papilla. DEVELOPMENT

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Fig. 5. Palatal Shh mRNA expression pattern in Pax9fneo/del;Wnt1Cre mutant embryos. (A-F) Patterns of Shh mRNA expression in the palate in wild-type (A-C) and Pax9fneo/del;Wnt1-Cre mutant (D-F) embryos from E12.5 to E14.5. p, palatal shelf; sp, sensory papilla.

Pax9 regulates mesenchymal-epithelial crosstalk during palate Whereas previous studies using section in situ hybridization analysis development. To investigate the molecular mechanism that mediates suggested that expression of both Msx1 and Bmp4 was restricted in Pax9 function in palate development, we carried out extensive the anterior palate (Zhang et al., 2002), our whole-mount in situ molecular marker analyses by using both quantitative real-time RT- hybridization assays demonstrated that, although Msx1 mRNA PCR and in situ hybridization analyses of E12.5 and E13.5 embryos. expression was restricted in the anterior palate (Fig. 8A-D), the Bmp4 Expression of Bmp4 and Msx1 mRNAs were consistently mRNA expression pattern in the developing palatal shelves consisted significantly reduced in the Pax9del/del mutant palate (Fig. 7A,B). of an anterior and a posterior domain separated by a Bmp4-negative middle palatal region (Fig. 8E,G). We further confirmed this unique pattern of Bmp4 mRNA expression by section in situ hybridization of the whole series of frontal sections through the developing palatal

Fig. 6. Disruption of the anterior-posterior boundary in the secondary palate in Pax9del/del mutant embryos. (A,B) Direct comparison of Shh (left half) and Shox2 (right half) mRNA expression patterns in the palatal shelf pairs in wild-type (A) and Pax9del/del mutant (B) embryos at E13.5. (C,D) Dual Fig. 7. Quantitative RT-PCR analysis of the levels of expression of Pax9, color in situ hybridization detection of Shh (brown) and Barx1 (turquoise) Msx1, Bmp4, Fgf10 and Osr2 mRNAs in E12.5 and E13.5 palatal shelves mRNAs in the palatal shelves of E13.5 wild-type (C) and Pax9del/del mutant in wild-type and Pax9del/del mutant embryos. (A) E12.5, (B) E13.5. Error

(D) embryos. bars represent s.e.m. *P<0.05; **P<0.01. DEVELOPMENT

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Fig. 8. Comparison of expression of Bmp4, Fgf10 and Msx1 mRNAs in the palatal shelves in Pax9del/del mutant and control embryos. (A-D) Whole-mount in situ hybridization results of Msx1 mRNA expression in wild-type (A,C) and Pax9del/del mutant (B,D) embryos at E12.5 (A,B) and E13.5 (C,D). (E-H) Whole-mount in situ hybridization results of Bmp4 mRNA expression in wild-type (E,G) and Pax9del/del mutant (F,H) embryos at E12.5 (E,F) and E13.5 (G,H). (I-N) Frontal sections showing expression of Bmp4 mRNAs in the anterior, middle and posterior regions of the developing palate in E13.5 wild-type (I,K,M) and Pax9del/del mutant (J,L,N) embryos. (O-T) Frontal sections showing expression of Fgf10 mRNA in the anterior, middle and posterior regions of the developing palate in E13.5 wild-type (O,Q,S) and Pax9del/del mutant (P,R,T) embryos. p, palatal shelf; t, tongue.

shelves of E13.5 mouse embryos and found that expression of Bmp4 palatal mesenchyme at E13.5 (Fig. 9C-H). Moreover, we found that mRNAs was significantly reduced in both the anterior and posterior expression of Fgf10 mRNAs was also downregulated in the regions of the developing palate of Pax9del/del mutant embryos in developing palatal shelves in Osr2−/− mutant embryos, in comparison comparison with control littermates (Fig. 8E-N). with wild-type littermates (Fig. 9I-L). Together, these results suggest Our real-time RT-PCR analyses also consistently detected a that Osr2 might act downstream of Pax9 to maintain Fgf10 expression reduction in the amount of Fgf10 mRNAs in the microdissected in the palatal mesenchyme during palatal outgrowth. E12.5 and E13.5 mutant palatal tissues in comparison with the control samples, although the difference was not statistically Expression of Osr2 from the Pax9 locus rescued posterior significant (Fig. 7A,B). This was probably due to the overall low palate morphogenesis in Pax9-deficient mice abundance and highly restricted domain of Fgf10 mRNA expression To further investigate the role of Osr2 in Pax9-mediated regulation in the developing palatal shelves, as Fgf10 mRNA expression was of palate development, we examined palatogenesis in the restricted to only the anterior and middle oral quarters of the Pax9Osr2KI/Osr2KI mice, in which an Osr2 cDNA replaced the first two developing palatal mesenchyme (Fig. 8O,Q,S). Indeed, section in exons of the endogenous Pax9 gene (Zhou et al., 2011). Whereas situ hybridization assays consistently showed reduced Fgf10 mRNA Pax9del/del mutant newborn pups had complete cleft secondary palate expression in the Pax9del/del mutant palate in comparison with (Fig. 10B), 50% of the Pax9Osr2KI/Osr2KI newborn pups exhibited control littermates (Fig. 8O-T). fused posterior secondary palate, with a partial cleft in the anterior We have previously shown that Pax9 is required for maintenance palate region only (n=24) (Fig. 10C). Histological analyses of E14.5 of Osr2 mRNA expression in the developing tooth mesenchyme embryos showed that the Pax9Osr2KI/Osr2KI mutant embryos exhibited (Zhou et al., 2011). In this study, we found that Osr2 mRNA rescue of the defects in palatal shelf elevation (n=4), in comparison expression was significantly reduced in the developing palate in the with the Pax9del/del mutant embryos that always showed defects in Pax9del/del mutant embryos, as detected by both real-time RT-PCR and palatal shelf elevation (Fig. 10D-F). By E16.5, 50% of the whole-mount in situ hybridization assays (Fig. 7A,B; Fig. 9A,B). We Pax9Osr2KI/Osr2KI mutant embryos showed a fused posterior palate further verified the changes in Osr2 mRNA expression by using (n=6) (Fig. 10L), but the anterior region of the palate shelves failed section in situ hybridization assay and found that Osr2 mRNA to make contact at the midline (Fig. 10I). expression was dramatically reduced in the middle and posterior Osr2 plays an important role in regulating palatal mesenchyme regions of the palatal shelves, whereas a higher level of Osr2 mRNA cell proliferation (Lan et al., 2004). We analyzed palatal cell del/del

expression was present in the very anterior region of the mutant proliferation in Pax9 embryos and control littermates by using DEVELOPMENT

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Fig. 9. Pax9 is required to maintain Osr2 mRNA expression in the developing palatal shelves. (A,B) Whole-mount in situ hybridization results of Osr2 mRNA expression in the palatal shelves in E12.5 wild-type control (A) and Pax9 mutant (B) littermates. (C-H) Frontal sections showing Osr2 mRNA expression in the anterior (C,D), middle (E,F) and posterior (G,H) regions of the developing palatal shelves in E13.5 wild-type (C,E,G) and Pax9del/del mutant (D,F,H) embryos. (I-L) Frontal sections showing Fgf10 mRNA expression in the anterior (I,J) and middle (K,L) regions of the developing palatal shelves in E13.5 wild-type (I,K) and Osr2−/− mutant (J,L) embryos . p, palatal shelf; t, tongue.

BrdU labeling and found that the posterior palate region, but not growth and indicating that the reduction in palatal growth the anterior palate region, had significant reduction in cell contributed to cleft palate pathogenesis in the Pax9del/del mice. proliferation in E13.5 Pax9del/del embryos in comparison with their We investigated further whether the rescued morphogenesis of the control littermates (Fig. 11A-D; Fig. 11I). This posterior palatal posterior palate in the Pax9Osr2KI/Osr2KI mutant embryos correlated growth defect was rescued in the Pax9Osr2KI/Osr2KI embryos with restoration of Bmp4, Msx1, Shh and Fgf10 mRNA expression (Fig. 11E-I), confirming a crucial role for Osr2 in palatal shelf during palate development. We examined more than four pairs of

Fig. 10. Partial rescue of palate morphogenesis in the Pax9Osr2KI/Osr2KI mutant mice. (A-C) Oral view of the palate in control (A), Pax9del/del mutant (B) and Pax9Osr2KI/Osr2KI mutant (C) newborn pups. In contrast to complete cleft secondary palate in the Pax9del/del mutant (asterisk in B), Pax9Osr2KI/Osr2KI mutant pups had a partial cleft of the anterior palate (rectangle in C). (D-F) Histology of frontal sections through the middle regions of the developing palate in E14.5 control (D), Pax9del/del mutant (E) and Pax9Osr2KI/Osr2KI mutant (F) embryos. Arrow in F indicates the midline epithelial seam of the palatal shelves. (G-L) Frontal sections through the anterior (G-I) and posterior (J-L) regions of the developing palate in E16.5 control (G,J), Pax9del/del mutant (H,K) and Pax9Osr2KI/Osr2KI mutant (I,L) embryos. Asterisk indicates the gap between the palatal shelves. p, palatal shelf; t, tongue. DEVELOPMENT

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Fig. 12. Comparison of patterns of expression of Msx1, Bmp4, Shh and Shox2 mRNAs in E13.5 wild-type and Pax9Osr2KI/Osr2KI mutant palate. (A,B) Whole-mount view of Msx1 mRNA expression in the palate in control (A) and Pax9Osr2KI/Osr2KI mutant (B) embryos. (C,D) Whole-mount view of Bmp4 mRNA expression in the palate in control (C) and Pax9Osr2KI/Osr2KI mutant (D) embryos. (E,F) Whole-mount in situ hybridization results for Shh (left half) and Shox2 (right half) mRNA expression in bisected upper jaw samples of control (E) and Pax9Osr2KI/Osr2KI mutant (F) embryos. R1 to R5 mark the rugae according to the temporal sequence of formation.

palate region, similar to the Pax9del/del mutant embryos (compare Fig. 12E,F and Fig. 4C,G). The domain of Shox2 mRNA expression Fig. 11. Analysis of cell proliferation in the developing palate in E13.5 del/del Osr2KI/Osr2KI in the palatal shelves was also similarly shifted anteriorly in the Pax9 and Pax9 embryos. (A-D) Representative images of Osr2KI/Osr2KI del/del sections through the anterior (A,B) and posterior (C,D) regions of the palate Pax9 embryos as in the Pax9 mutant embryos in Pax9del/+ control (A,C) and Pax9del/del mutant (B,D) embryos showing (compare Fig. 12E,F and Fig. 6A,B). However, we found that two Osr2KI/Osr2KI distribution of immunostained BrdU-labeled nuclei (red). Sections were out of five Pax9 mutant embryos had restored Fgf10 counterstained with Hoechst (blue). (E-H) Representative images of sections mRNA expression in the anterior and middle palate regions at E13.5 through the anterior (E,F) and posterior (G,H) regions of the palate in (Fig. 13A-D). Together, these results suggest that Osr2 acts Osr2KI/+ Osr2KI/Osr2KI Pax9 (E,G) and Pax9 mutant (F,H) embryos showing downstream of or in parallel with the Bmp4 and Shh pathways and distribution of immunostained BrdU-labeled nuclei (red). White line indicates cooperates with Pax9 to maintain Fgf10 expression during palate approximate position below which the palatal mesenchyme cells were counted. (I) The percentage of BrdU-labeled cells in the E13.5 palatal development. mesenchyme. Error bar represents s.d. *P<0.01. DISCUSSION Although cleft palate in Pax9-deficient mice was first reported E13.5 embryos for each probe and all Pax9Osr2KI/Osr2KI mutant more than 15 years ago (Peters et al., 1998), the molecular embryos exhibited reduced expression of Msx1 and Bmp4 mRNAs mechanisms involving Pax9 in palate development have not been in the developing palatal shelves, in comparison with the wild-type documented. In this study, we found that Pax9 regulates littermates (Fig. 12A-D). Moreover, the Pax9Osr2KI/Osr2KI mutant mesenchymal-epithelial signaling and AP patterning during palate embryos showed loss of Shh mRNA expression in the posterior outgrowth and that Pax9 acts upstream of Osr2 to regulate palatal

palate and decreased Shh mRNA expression in the rugae in the mid- shelf growth and elevation. DEVELOPMENT

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palatal mesenchyme and that Bmp4 expression in the posterior palate may also be involved in maintenance of Shh expression in the palatal epithelium. In addition to Bmp4, mesenchymally expressed Fgf10 is involved in maintenance of Shh expression in the palatal epithelium, with Fgf10 and Shh acting in a positive-feedback loop to regulate palatal outgrowth (Lan and Jiang, 2009; Rice et al., 2004). Fgf10 mRNA expression was downregulated in the Pax9del/del mutant palatal shelves. Interestingly, we found that Fgf10 mRNA expression was also downregulated in the Osr2−/− mutant palatal shelves and that Osr2 mRNA expression was significantly downregulated in Pax9del/del mutant palatal shelves. Moreover, we found that Fgf10 mRNA expression was restored in a subset of Pax9Osr2KI/Osr2KI mutant palate, which correlated with partial rescue of palate morphogenesis in the Pax9Osr2KI/Osr2KI mutant embryos. Together, these data suggest that Pax9 may regulate Fgf10 expression in the Fig. 13. Comparison of Fgf10 mRNA expression in the developing developing palatal mesenchyme through Osr2. −/− del/del palate in E13.5 control and Pax9Osr2KI/Osr2KI mutant embryos. Whereas Osr2 and Pax9 mutant mice each had cleft palate, (A-D) Representative frontal sections through the anterior (A,B) and middle the palatal were not the same. Osr2−/− mutant embryos (C,D) regions of the developing palatal shelves were used for detection of had reduced mesenchyme cell proliferation throughout the AP axis Fgf10 mRNA expression by section in situ hybridization. p, palatal shelf; t, of the palatal shelves at E13.5 (Lan et al., 2004), whereas Pax9del/del tongue. mutant embryos showed reduced mesenchymal cell proliferation only in the posterior region and exhibited malformed palatal shelves Reciprocal signaling between the epithelium and neural crest lacking the lateral indentation. However, both Osr2−/− and Pax9del/del derived mesenchyme plays a crucial role in the growth of the mutant embryos had defects in palatal shelf elevation (Lan et al., secondary palate (Bush and Jiang, 2012; Lan and Jiang, 2009; Rice 2004; and this study). We found that Osr2 mRNA expression was et al., 2004; Welsh and O’Brien, 2009). We found that disruption of significantly downregulated in Pax9del/del mutant palatal shelves as patterns of Shh expression in the palatal epithelium was among the early as E12.5 and that restoration of Osr2 expression in the palatal earliest detectable defects during palate development in the mesenchyme through the Pax9Osr2KI allele rescued palatal elevation Pax9del/del mutant mice. Remarkably, Shh mRNA expression in the and posterior palate morphogenesis in ~50% of the Pax9Osr2KI/Osr2KI palatal epithelium was also disrupted in the Pax9fNeo/delWnt1-Cre mutant mice (Fig. 10). Whether Osr2 is a direct target of Pax9 mutant embryos (Fig. 5), indicating that Pax9-mediated transcriptional regulation requires further investigation, however, transcriptional regulation in the neural crest-derived palatal because maintenance of Osr2 mRNA expression in the developing mesenchyme indirectly regulates Shh expression in the palatal palatal shelves requires active Shh signaling (Lan and Jiang, 2009) epithelium. Previous studies of cleft palate pathogenesis in the but Shh expression was significantly perturbed in the Pax9del/del Msx1−/− mutant mice implicated a role for Bmp4 acting downstream mutant palate. Moreover, although posterior palate morphogenesis of Msx1 in the palatal mesenchyme but upstream of Shh expression was rescued in the Pax9Osr2KI/Osr2KI embryos in comparison with in the palatal epithelium in the anterior palate (Zhang et al., 2002). Pax9del/del mutants, the restoration of Osr2 to the palatal Whereas Zhang et al. (Zhang et al., 2002), using only the section in mesenchyme through the Pax9Osr2KI allele was insufficient to rescue situ hybridization assay, suggested that expression of both Msx1 and the effects of loss of Pax9 on expression of Bmp4, Msx1 and Shh in Bmp4 was restricted to the anterior palate region, our whole-mount the developing palate. Furthermore, we found that Osr2 mRNA in situ hybridization assays clearly detected strong Bmp4 mRNA expression was more dramatically reduced in the middle and expression in a discrete domain in the posterior palate at both E12.5 posterior regions of the Pax9del/del mutant palate, which correlated and E13.5 (Fig. 8E,G). Levi et al. (Levi et al., 2006) reported whole- with the reduced expression of Shh mRNA expression in the middle mount in situ hybridization results for Bmp4 mRNA expression in and posterior regions of the palatal epithelium in these mutant the E13.5 palate and also clearly showed a strong Bmp4 expression embryos. Together, these results suggest that Pax9 plays a primary domain in the posterior palate (Levi et al., 2006). We carried out role in regulating the mesenchymal-epithelial interactions involving section in situ hybridization of the whole series of frontal sections the Bmp4 and Shh signaling, and that Osr2 acts downstream of these of E13.5 mouse palate and confirmed the separate Bmp4 mRNA pathways to regulate palatal cell proliferation and palatal shelf expression domains in the anterior and posterior regions of the elevation. palatal shelves (Fig. 8I,K,M). Moreover, Levi et al. (Levi et al., Defects in palatal shelf elevation have been attributed to 2006) showed that Bmp4 mRNA expression in the anterior palate, malformation of or physical obstruction by the tongue in several but not in the posterior palate, was reduced in the Msx1−/− embryos mutant mouse strains (Barrow et al., 2000; Dudas et al., 2004; in comparison with wild-type control embryos (Levi et al., 2006). Gendron-Maguire et al., 1993; Murray et al., 2007; Wang et al., 2003). We found that the posterior domain of Bmp4 mRNA expression was In the Pax9del/del mutant embryos, failure of palatal shelf elevation was significantly downregulated in the Pax9del/del mutant embryos, also accompanied by abnormal morphology of the tongue indicating that Pax9 regulates Bmp4 expression in the posterior (Fig. 2K,L,R). However, we found that Pax9del/del mutant embryos had palate independently of Msx1. Previous biochemical studies have defects in expression of Shh, Msx1 and Bmp4 by E12.5, and exhibited shown that Pax9 could activate the luciferase reporter expression abnormally broadened mid-palate region by E13.5, indicating that the driven by either the Msx1 or Bmp4 gene promoter in co-transfected mutant palatal shelves were already malformed 1 day before the time COS7 cells (Ogawa et al., 2006). Together, these data suggest that of normal palatal shelf elevation. The abnormal broadening of the

Pax9 directly regulates both Msx1 and Bmp4 expression in the palatal shelves in the mid-palate region correlated with the disruption DEVELOPMENT

4716 RESEARCH ARTICLE Development (2013) doi:10.1242/dev.099028 of the anterior-posterior boundary of the palatal shelves, whereas the in paraffin and sectioned at 7 μm. Whole-mount (Baek et al., 2011; Lan et lack of indentation lateral to the palatal shelves correlated with the al., 2001) and section in situ hybridization (Zhang et al., 1999) were carried early developmental arrest of the molar tooth germs in the mutant out as described previously with digoxygenin or fluorescein-labeled embryos. As all Pax9Osr2KI/Osr2KI embryos also had early antisense RNA probes. For section in situ hybridization, frontal sections developmental arrest of the molar tooth germs (Zhou et al., 2011), but through palatal shelves were selected from the anterior, middle and posterior regions, with the middle region consisting of sections through the maxillary about half of those mutants developed fused posterior secondary first molar tooth buds. palate (Fig. 10L), the tooth developmental defect was unlikely to be For histology, embryos were fixed in either Bouin’s fixative or 4% a determining factor in palatal shelf elevation. However, we found that paraformaldehyde. Fixed embryos were dehydrated through a graded series Osr2KI/Osr2KI some, but not all, of the Pax9 embryos had restored Fgf10 of ethanol and embedded in paraffin wax. Serial sections of 7 μm were mRNA expression in the palatal mesenchyme, which correlated with stained with Hematoxylin and Eosin. partial rescue of palate morphogenesis in those mutants. Thus, although we have not ruled out possible contribution by abnormalities BrdU labeling and cell proliferation assay of surrounding structures, including the tooth germs and tongue, to the For detection of cell proliferation in the palatal shelves, timed-mated impairment of palatal shelf elevation in the Pax9del/del mutant embryos, pregnant female mice were injected once intraperitoneally at gestational day our data support a crucial role for Pax9-dependent gene expression 13.5 with the BrdU Labeling Reagent (Roche, 15 μl/g body weight). One within the developing palatal shelves in palatal shelf elevation. hour after injection, embryos were dissected, fixed in 4% paraformaldehyde Osr2KI/Osr2KI at 4°C overnight, embedded in paraffin wax and sectioned at 5 μm in the Whereas 50% of late-term Pax9 embryos showed coronal plane. The sections were rehydrated and submerged in pre-cooled fusion of the posterior palate, all of the mutant embryos had cleft of acetone at −20°C for 30 minutes. After washing with PBS, the specimens the anterior palate. The anterior cleft palate defect correlated with were treated with 20 μg/ml proteinase K at room temperature for 10 the reduction in Msx1 expression in the anterior palate in both minutes. After washing with PBS, sections were treated with 2 M HCl for Pax9del/del and Pax9Osr2KI/Osr2KI embryos (Fig. 12C,D). As Msx1 is 1 hour at room temperature to denature the genomic DNA. Samples were not expressed in the posterior palate, these results indicate that Pax9 rinsed in PBS for three times, then incubated in blocking solution (2% BSA, regulates palate development through distinct molecular pathways 10% goat serum, 0.1% Tween in PBS) at room temperature for 30 minutes, in the anterior and posterior regions. Similar to the anterior palatal followed by incubation with Alexa Fluor 594-conjugated anti-BrdU mesenchyme, the developing tooth bud mesenchyme exhibits antibody (Invitrogen, #B35132) solution (1:50 in blocking solution) at 4°C overnight in a humid atmosphere. The sections were rinsed in PBS, partially overlapping expression of Msx1, Pax9 and Osr2 (Zhang et counterstained with Hoechst and mounted with anti-fade mounting gel. al., 2009; Zhou et al., 2011). We recently discovered that Msx1 and Images were obtained by using a Zeiss fluorescence microscope and Osr2 interact physically but act antagonistically in the regulation of analyzed with Imaris software. The numbers of BrdU-labeled nuclei and mesenchymal odontogenic activity (Jia et al., 2013; Zhang et al., total nuclei were recorded for two independent control and mutant littermate 2009; Zhou et al., 2011). It is possible that the Msx1-Osr2 pairs. The cell proliferation data were recorded and analyzed separately for interaction may play an important role in the development of the the anterior and posterior regions of the palatal shelves, with data from 15- anterior palate. In the Pax9Osr2KI/Osr2KI embryos, the reduction of 20 sections from each region of each palatal shelf. Student’s t-test was used Msx1 expression affects the pathways downstream of Msx1-Osr2 to analyze the differences in the datasets and P<0.01 was considered interactions, which would account for the anterior cleft palate defect. statistically significant. Together, these results indicate that both Osr2 and Pax9 interact with the distinct molecular pathways in the anterior and posterior palate. Real-time RT-PCR Osr2KI/Osr2KI Palatal shelves were micro-dissected from E12.5 and E13.5 embryos, The Pax9 mutant mice provide a valuable tool for quickly frozen in liquid nitrogen and stored individually at −80°C. comprehensive analysis of the gene regulatory network controlling Following identification of the genotypes by allele-specific PCR, two pairs palate development and patterning. of palatal shelf samples were pooled by genotype. Total RNA was extracted using the RNeasy Micro Kit (Qiagen). First-strand cDNA synthesis was MATERIALS AND METHODS achieved using the SuperScript First-Strand Synthesis System (Invitrogen). Mouse strains Quantitative PCR amplifications were performed in a C1000 Touch Pax9fneo/+, Pax9del/+ and Pax9Osr2KI/+ mice have been described previously Thermal Cycler (Bio-Rad) using the SYBR GreenER qPCR Supermix (Zhou et al., 2011). Pax9del/+ and Pax9Osr2KI/+ mice were maintained in the (Invitrogen). Gene-specific primers were: Hprt-F (5′-TGCTGGT- C57BL/6J strain background and were intercrossed to generate homozygous GAAAAGGACCTCTCG-3′) and Hprt-R (5′-CTGGCAACAT- mutants for analyses. Pax9fneo/+ mice were crossed with Wnt1-Cre transgenic CAACAGGACTCC-3′); Pax9-F (5′-TATTCTGCGCAACAAGATCG-3′) mice (Danielian et al., 1998) to generate Pax9fneo/+Wnt1-Cre mice, which and Pax9-R (5′-GGTGGTGTAGGCACCTTAGC-3′); Bmp4-F (5′- were then crossed with Pax9del/+ mice to generate Pax9fneo/delWnt1-Cre GAGGGATCTTTACCGGCTCC-3′) and Bmp4-R (5′-GTTGAAGAG- mutant embryos for analyses. GAAACGAAAAGCAG-3′); Msx1-F (5′-AAGATGCTCTGGTGAAGGC- 3′) and Msx1-R (5′-TGGTCTTGTGCTTGCGTA-3′); Fgf10-F (5′- Scanning electron microscopy TTTGAGCCATAGAGTTTCCCC-3′) and Fgf10-R (5′-CGGGACCAA- Embryos were fixed with 2% glutaraldehyde/2% paraformaldehyde in PBS GAATGAAGACTG-3′); Osr2-F (5′-TCTTTACACATCCCGCTTCC-3′) and Osr2-R (5′-GGAAAGGTCATGAGGTCCAA-3′). For each sample, the followed by OsO4 treatment and dehydration through an ethanol series. The relative levels of target mRNAs were normalized to that of HPRT using the embryos were critical point dried under CO2 and sputter coated with 30 nm gold particles. Images were obtained using Zeiss Supra 40VP Field Emission standard curve method. Four sets of samples were analyzed for each gene. at the Electron Microscope Research Core of the University of Rochester Student’s t-test was used to analyze the difference and P<0.05 was Medical Center (NY, USA). considered statistically significant.

Acknowledgements Histology and in situ hybridization We thank Samantha Brugmann, Yang Chai and Steven Potter for comments and Embryos were collected at desired developmental stages and fixed in 4% suggestions. paraformaldehyde overnight at 4°C. For whole-mount in situ hybridization, embryos were dehydrated through graded methanol. For section in situ Competing interests

hybridization, embryos were dehydrated through graded alcohols, embedded The authors declare no competing financial interests. DEVELOPMENT

4717 RESEARCH ARTICLE Development (2013) doi:10.1242/dev.099028

Author contributions Lan, Y., Ovitt, C. E., Cho, E. S., Maltby, K. M., Wang, Q. and Jiang, R. (2004). Odd- Y.L. and R.J. conceived and designed this research project. J.Z., Y.G., Y.L. and skipped related 2 (Osr2) encodes a key intrinsic regulator of secondary palate S.J. carried out the experiments. All authors participated in analysis and discussion growth and morphogenesis. Development 131, 3207-3216. of the experimental data. J.Z. and Y.G. wrote the initial draft of the manuscript. All Levi, G., Mantero, S., Barbieri, O., Cantatore, D., Paleari, L., Beverdam, A., authors participated in editing and finalizing the manuscript. Genova, F., Robert, B. and Merlo, G. R. (2006). Msx1 and Dlx5 act independently in development of craniofacial skeleton, but converge on the regulation of Bmp signaling in palate formation. Mech. Dev. 123, 3-16. Funding Li, Q. and Ding, J. (2007). Gene expression analysis reveals that formation of the This work was supported by National Institutes of Health (NIH)/National Institute of mouse anterior secondary palate involves recruitment of cells from the posterior Dental and Craniofacial Research (NIDCR) [DE013681 and DE015207 to R.J.]. side. Int. J. Dev. Biol. 51, 167-172. Deposited in PMC for release after 12 months. Liu, W., Lan, Y., Pauws, E., Meester-Smoor, M. A., Stanier, P., Zwarthoff, E. C. and Jiang, R. (2008). The Mn1 transcription factor acts upstream of Tbx22 and References preferentially regulates posterior palate growth in mice. Development 135, 3959- Alappat, S. R., Zhang, Z., Suzuki, K., Zhang, X., Liu, H., Jiang, R., Yamada, G. and 3968. Chen, Y. (2005). The cellular and molecular etiology of the cleft secondary palate in Murray, S. A., Oram, K. F. and Gridley, T. (2007). Multiple functions of Snail family Fgf10 mutant mice. Dev. Biol. 277, 102-113. genes during palate development in mice. Development 134, 1789-1797. Baek, J. A., Lan, Y., Liu, H., Maltby, K. M., Mishina, Y. and Jiang, R. (2011). Bmpr1a Ogawa, T., Kapadia, H., Feng, J. Q., Raghow, R., Peters, H. and D’Souza, R. N. signaling plays critical roles in palatal shelf growth and palatal bone formation. Dev. (2006). Functional consequences of interactions between Pax9 and Msx1 genes in Biol. 350, 520-531. normal and abnormal tooth development. J. Biol. Chem. 281, 18363-18369. Barlow, A. J., Bogardi, J. P., Ladher, R. and Francis-West, P. H. (1999). Expression Pantalacci, S., Prochazka, J., Martin, A., Rothova, M., Lambert, A., Bernard, L., of chick Barx-1 and its differential regulation by FGF-8 and BMP signaling in the Charles, C., Viriot, L., Peterkova, R. and Laudet, V. (2008). Patterning of palatal maxillary primordia. Dev. Dyn. 214, 291-302. rugae through sequential addition reveals an anterior/posterior boundary in palatal Barrow, J. R., Stadler, H. S. and Capecchi, M. R. (2000). Roles of Hoxa1 and Hoxa2 development. BMC Dev. Biol. 8, 116. in patterning the early hindbrain of the mouse. Development 127, 933-944. Peters, H., Neubüser, A., Kratochwil, K. and Balling, R. (1998). Pax9-deficient mice Bush, J. O. and Jiang, R. (2012). Palatogenesis: morphogenetic and molecular lack pharyngeal pouch derivatives and teeth and exhibit craniofacial and limb mechanisms of secondary palate development. Development 139, 231-243. abnormalities. Genes Dev. 12, 2735-2747. Coulter, D. E. and Wieschaus, E. (1988). Gene activities and segmental patterning in Rice, R., Spencer-Dene, B., Connor, E. C., Gritli-Linde, A., McMahon, A. P., Drosophila: analysis of odd-skipped and pair-rule double mutants. Genes Dev. 2, Dickson, C., Thesleff, I. and Rice, D. P. (2004). Disruption of Fgf10/Fgfr2b- 1812-1823. coordinated epithelial-mesenchymal interactions causes cleft palate. J. Clin. Invest. Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K. and McMahon, A. P. 113, 1692-1700. (1998). Modification of gene activity in mouse embryos in utero by a tamoxifen- Rice, R., Connor, E. and Rice, D. P. (2006). Expression patterns of Hedgehog inducible form of Cre recombinase. Curr. Biol. 8, 1323-1326. signalling pathway members during mouse palate development. Gene Expr. Dudas, M., Sridurongrit, S., Nagy, A., Okazaki, K. and Kaartinen, V. (2004). Patterns 6, 206-212. Craniofacial defects in mice lacking BMP type I Alk2 in neural crest cells. Saulier-Le Dréan, B., Nasiadka, A., Dong, J. and Krause, H. M. (1998). Dynamic Mech. Dev. 121, 173-182. changes in the functions of Odd-skipped during early Drosophila embryogenesis. Ferguson, M. W. (1988). Palate development. Development 103 Suppl., 41-60. Development 125, 4851-4861. Gendron-Maguire, M., Mallo, M., Zhang, M. and Gridley, T. (1993). Hoxa-2 mutant Stapleton, P., Weith, A., Urbánek, P., Kozmik, Z. and Busslinger, M. (1993). mice exhibit homeotic transformation of skeletal elements derived from cranial neural Chromosomal localization of seven and cloning of a novel family crest. Cell 75, 1317-1331. member, PAX-9. Nat. Genet. 3, 292-298. Green, R. B., Hatini, V., Johansen, K. A., Liu, X. J. and Lengyel, J. A. (2002). Wang, L. and Coulter, D. E. (1996). bowel, an odd-skipped homolog, functions in the Drumstick is a protein that antagonizes Lines to control patterning and terminal pathway during Drosophila embryogenesis. EMBO J. 15, 3182-3196. morphogenesis of the Drosophila . Development 129, 3645-3656. Wang, T., Tamakoshi, T., Uezato, T., Shu, F., Kanzaki-Kato, N., Fu, Y., Koseki, H., Gritli-Linde, A. (2007). Molecular control of secondary palate development. Dev. Biol. Yoshida, N., Sugiyama, T. and Miura, N. (2003). Forkhead transcription factor 301, 309-326. Foxf2 (LUN)-deficient mice exhibit abnormal development of secondary palate. Dev. Hao, I., Green, R. B., Dunaevsky, O., Lengyel, J. A. and Rauskolb, C. (2003). The odd-skipped family of zinc finger genes promotes Drosophila leg segmentation. Dev. Biol. 259, 83-94. Biol. 263, 282-295. Welsh, I. C. and O’Brien, T. P. (2009). Signaling integration in the rugae growth zone Hart, M. C., Wang, L. and Coulter, D. E. (1996). Comparison of the structure and directs sequential SHH signaling center formation during the rostral outgrowth of the expression of odd-skipped and two related genes that encode a new family of zinc palate. Dev. Biol. 336, 53-67. finger in Drosophila. Genetics 144, 171-182. Yu, L., Gu, S., Alappat, S., Song, Y., Yan, M., Zhang, X., Zhang, G., Jiang, Y., Hilliard, S. A., Yu, L., Gu, S., Zhang, Z. and Chen, Y. P. (2005). Regional regulation of Zhang, Z., Zhang, Y. et al. (2005). Shox2-deficient mice exhibit a rare type of palatal growth and patterning along the anterior-posterior axis in mice. J. Anat. 207, incomplete clefting of the secondary palate. Development 132, 4397-4406. 655-667. Zhang, Y., Zhao, X., Hu, Y., St Amand, T., Zhang, M., Ramamurthy, R., Qiu, M. and Ito, Y., Yeo, J. Y., Chytil, A., Han, J., Bringas, P., Jr, Nakajima, A., Shuler, C. F., Chen, Y. (1999). Msx1 is required for the induction of Patched by Sonic hedgehog in Moses, H. L. and Chai, Y. (2003). Conditional inactivation of Tgfbr2 in cranial neural the mammalian tooth germ. Dev. Dyn. 215, 45-53. crest causes cleft palate and calvaria defects. Development 130, 5269-5280. Zhang, Z., Song, Y., Zhao, X., Zhang, X., Fermin, C. and Chen, Y. (2002). Rescue of Jia, S., Zhou, J., Gao, Y., Baek, J. A., Martin, J. F., Lan, Y. and Jiang, R. (2013). cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP Roles of Bmp4 during tooth morphogenesis and sequential tooth formation. and Shh signaling in the regulation of mammalian palatogenesis. Development 129, Development 140, 423-432. 4135-4146. Lan, Y. and Jiang, R. (2009). Sonic hedgehog signaling regulates reciprocal epithelial- Zhang, Z., Lan, Y., Chai, Y. and Jiang, R. (2009). Antagonistic actions of Msx1 and mesenchymal interactions controlling palatal outgrowth. Development 136, 1387-1396. Osr2 pattern mammalian teeth into a single row. Science 323, 1232-1234. Lan, Y., Kingsley, P. D., Cho, E. S. and Jiang, R. (2001). Osr2, a new mouse gene Zhou, J., Gao, Y., Zhang, Z., Zhang, Y., Maltby, K. M., Liu, Z., Lan, Y. and Jiang, R. related to Drosophila odd-skipped, exhibits dynamic expression patterns during (2011). Osr2 acts downstream of Pax9 and interacts with both Msx1 and Pax9 to craniofacial, limb, and kidney development. Mech. Dev. 107, 175-179. pattern the tooth developmental field. Dev. Biol. 353, 344-353. DEVELOPMENT

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